Dissecting the cell of origin of aberrant SALL4 expression in myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a group of heterogeneous diseases characterized by cytologic dysplasia and cytopenias resulting from ineffective hematopoiesis. Oncofetal protein SALL4 is a known oncogene in MDS and its baseline expression level serves as a prognostic biomarker for MDS at the time of diagnosis. In addition, a recent study showed that SALL4 upregulation following hypomethylating agent treatment in MDS patients correlates with poor outcomes. Despite its important mechanistic and diagnostic significance, the cellular identity of bone marrow cells with aberrant SALL4 expression in MDS patients remains unknown. In this study, we analyzed MDS bone marrow cells on single cell level by mass cytometry (CyTOF) and found that SALL4 was mainly aberrantly expressed in the hematopoietic stem and progenitor cells (HSPC) as well as myeloid lineages. Within the HSPC population from MDS patients, SALL4 and p53 were co-expressed, with the highest co-expressing clones harboring pathogenic TP53 mutations. Overall, our study characterizes for the first time the aberrant SALL4 expression in primary MDS patient samples at a single-cell level. Further studies on the SALL4/p53 network for in-depth mechanistic investigation are needed in the future. Key Points SALL4 expression in various MDS BM cells confirmed by mass cytometry (CyTOF). SALL4 and p53 double positive cells were predominantly found in the hematopoietic stem and progenitor cell (HSPC) population and associated with pathogenic TP53 mutation status.

to 30% of patients with MDS may transform to acute myeloid leukemia (AML). Despite the well-characterized cytogenetic changes and gene mutations associated with MDS, the pathogenesis of the disease remains incompletely understood, which is a major barrier to developing effective therapeutic strategies. 1 SALL4, a transcription factor important for development and embryonic stem cell properties, 2 is aberrantly expressed in various cancers 3,4 and is a known leukemic oncogenic driver. [5][6][7] Its expression level serves as a prognostic biomarker for MDS at the time of diagnosis. 8 We recently showed that SALL4 upregulation following hypomethylating agent treatment in MDS patients correlates with poor outcomes. 9 To further investigate the cellular identity of bone marrow cells with aberrant SALL4 expression in MDS patients, we utilized mass cytometry (CyTOF) to analyze MDS bone marrow cells in comparison to controls. Additionally, to address the relationship between SALL4 expression and somatic gene mutations, which is important for risk stratification and identification of disease drivers, we performed paired bone marrow Whole Exome Sequencing (WES) on these samples. DNA from bone marrow (BM) mononuclear cells from MDS and control samples (lymphoma without bone marrow infiltration) were extracted according to the manufacturer's recommendations (Table S1). We first examined SALL4 mRNA expression in MDS CD34+ cells by analyzing MDS expression profiles from the public database GSE19429, which contained 183 patients with MDS and 17 healthy controls. We observed that SALL4 expression in MDS CD34 cells was higher than that of controls, with the highest expression in refractory anemia with excess blasts type 2 (RAEB2) (p ≤ .05) ( Figure 1A,B). To further investigate the protein expression pattern of SALL4 in MDS patients' BM, we performed CyTOF experiments, which included 22 surface markers of cell surface proteins and 5 markers of intracellular signaling with single-cellular resolution (Table S2). We validated the CyTOF method in cell lines, using published gating methods ( Figures S1 and S2). Our results showed that MDS patients had aberrant SALL4 protein expression in hematopoietic stem cells (HSC) (p ≤ .05), hematopoietic progenitor cells (HPC) (p ≤ .05) and myeloid lineage cells (p ≤ .05) when compared to controls ( Figure 1C and Figure  S2). Understanding the cell of origin with aberrant SALL4 expression has important implications for understanding MDS pathogenesis, as well as SALL4 therapeutic targeting in myeloid neoplasms. Recent advances in the understanding of myeloid neoplasm pathogenesis and refined analysis of AML bone marrow cell-of-origin studies have led to the design of therapeutic targeting of leukemia stem cells as a more promising approach toward a cure.
Next, we investigated the relationship between SALL4 expression with p53, ki67, c-myc or phosphorylated AKT (pAKT), which have important roles in leukemia or MDS pathogenesis, 1 on single cell level. SALL4 expressing cells expressed p53 (p = .01) in hematopoietic stem/progenitor (HSPC) (Figure 2A) and showed a trend of a higher level of pAKT and ki67.
Next, FlowSOM via viSNE analysis was performed on MDS bone marrow cells to understand the expression pattern of SALL4 and p53 and to investigate the relationship between them. This analysis distinguished 49 clusters ( Figure 2B & C). SALL4 expressing clusters tended to express p53, whereas SALL4 non-expressing cells did not ( Figure 2C & D). Minimal spanning trees (MSTs) analysis by FlowSOM ( Figure 3A) distinguished 10 meta-clusters ( Figure 3B, Figure S3A), with SALL4+p53+ cells mainly present in meta-cluster 9 ( Figure 3C, Figures S3 and S4).
Previously, the relationship between SALL4 expression and somatic gene mutations (e.g., TP53), which are important for risk stratification and identification of disease drivers, had not been well explored in MDS patients. Using concomitant WES data, we were able to gain further insights into the relationship between the TP53 mutation status and SALL4/TP53 co-expression in MDS patients. Notably, both patients number 1 and number 2, who had the highest fractions of SALL4/TP53 double positive cells, harbored pathogenic loss-of-function TP53 mutations in the DNA binding domain (Table 1). For patient NO.7, the p.V173* mutation was a frameshift variant that likely resulted in nonsense mediated decay and reduced/absent TP53 protein expression. Thus, it was not surprising that a significantly smaller fraction of SALL4/TP53 positive cells was detected in patient NO.7 despite the presence of a deleterious TP53 mutation. Using the TCGA AML dataset, we found that TP53 mutations are enriched in patients having high SALL4 expression ( Figure S5). The mechanistic interplay between SALL4 and TP53 requires further investigation.

CONCLUSION
The identification of MDS-clones in MDS patients that may progress to AML is of great importance for the treatment of this disease. While SALL4, when constitutively expressed, has been shown to cause MDS/AML in a murine model, the cellular identity of aberrant SALL4 in human MDS remains unknown. Utilizing stateof-the-art single cell mass cytometry and paired Whole Exome Sequencing, our study has, for the first time, identified aberrant expression of SALL4 in hematopoietic stem/progenitor cells and myeloid lineage cells in human MDS patient bone marrow samples. Additionally, we observed a significant SALL4+p53+ cluster in the MDS bone marrow, particularly prominent in patients with pathogenic TP53 mutations. Our study provides a foundation for future investigations into the mechanisms and therapeutic targeting of SALL4 in the pathologic lineages of MDS, with potential prognostic and therapeutic implications.